Tolerance and dose-response assessment of subchronic dietary ethoxyquin exposure in Atlantic salmon (Salmo salar L.)

Ethoxyquin (EQ; 6-Ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed components for pets, livestock and aquaculture. However, possible risks of EQ used in aquafeed for fish health have not yet been characterized. The present study investigated the toxicity and dose-response of subchronic dietary EQ exposure at doses ranging from 41 to 9666 mg EQ/kg feed in Atlantic salmon (Salmo salar L.). Feed at concentrations higher than 1173 mg EQ/kg were rejected by the fish, resulting in reduced feed intake and growth performance. No mortality was observed in fish exposed to any of the doses. A multi-omic screening of metabolome and proteome in salmon liver indicated an effect of dietary EQ on bioenergetics pathways and hepatic redox homeostasis in fish fed concentrations above 119 mg EQ/kg feed. Increased energy expenditure associated with an upregulation of hepatic fatty acid â-oxidation and induction and carbohydrate catabolic pathways resulted in a dose-dependent depletion of intracytoplasmic lipid vacuoles in liver histological sections, decreasing whole body lipid levels and altered purine/pyrimidine metabolism. Increased GSH and TBARS in the liver indicated a state of oxidative stress, which was associated with activation of the NRF2-mediated oxidative stress response and glutathione-mediated detoxification processes. However, no oxidative DNA damage was observed. As manifestation of altered energy metabolism, the depletion of liver intracytoplasmic lipid vacuoles was considered the critical endpoint for benchmark dose assessment, and a BMDL10 of 243 mg EQ/kg feed was derived as a safe upper limit of EQ exposure in Atlantic salmon

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting.publishedVersio

Evaluation of an FIA Operated Amperometric Bacterial Biosensor, Based on Pseudomonas Putida F1 for the Detection of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX)

Recently, the development and optimization of a flow injection analysis (FIA) operated bacterial biosensor based on the aerobic catabolism of Pseudomonas putida ML2 was reported in the literature (Lanyon et al. 2004, 2005). By adapting information from these reports, we investigated whether operating parameters and procedures of the benzene biosensor could be directly applied to a new system based on a different bacterial strain for the detection of the whole benzene, toluene, ethylbenzene, and xylenes range. Cells of the investigated bacterial strain, Pseudomonas putida F1, were immobilized between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. The P. putida F1 aerobically degrades benzene, toluene, and ethylbenzene (BTE) (Cho et al. 2000). The BTE biosensor in kinetic mode FIA displayed a linear range of 0.02-0.14 mM benzene (response time: 5 min, base-line recovery time: 15 min), 0.05-0.2 mM toluene (response time: 8 min, baseline recovery time: 20 min), and 0.1-0.2 mM ethylbenzene (response time: 12 min, baseline recovery time: 30 min), respectively. Due to the differences in sensitivity, response, and baseline recovery times for BTE, it was possible to differentiate each compound in mixtures of these volatile organic compounds (VOCs). No response for xylenes could be obtained since they cannot be completely metabolized by this bacterial strain. However, it was reported that the range of compounds degradable by P. putida F1 can possibly be expanded by cultivating the cells on different carbon sources (Choi et al. 2003). The sensor showed good intra- and interassay reproducibility, and all obtained results were comparable with those reported in the literature. The demonstrated reproducibility and the simplicity and ease of use as well as the portability for in situ measurements indicates that the biosensor could be suitable as a reliable initial warning device for elevated BTE levels in indoor and outdoor environments

compareMS2 2.0: An Improved Software for Comparing Tandem Mass Spectrometry Datasets.

It has long been known that biological species can be identified from mass spectrometry data alone. Ten years ago, we described a method and software tool, compareMS2, for calculating a distance between sets of tandem mass spectra, as routinely collected in proteomics. This method has seen use in species identification and mixture characterization in food and feed products, as well as other applications. Here, we present the first major update of this software, including a new metric, a graphical user interface and additional functionality. The data have been deposited to ProteomeXchange with dataset identifier PXD034932.publishedVersio

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting